A family of Dual Oxidases (Duox) has been defined. Duox enzymes consist of three domains: 1) an NADPH-oxidase domain homologous to gp91phox, the catalytic subunit of the phagocyte respiratory burst oxidase; 2) a calcium-binding domain; and 3) a peroxidase domain. Located in plasma membrane of epithelial cells (colon, lung, thyroid, pancreas), Duoxs' are proposed to function by using intracellular NADPH to reduce 02 to form H202 outside the cell. The latter is used by the peroxidase domain to catalyze peroxidative reactions involving modification of extracellular matrix proteins and probably other extracellular small molecules. Specifically, Duox's are proposed to function biologically in innate immunity, endocrine function and cancer. We identified and cloned two human isoforms, h-Duox1 and h-Duox2, and have identified Duox enzymes in diverse species, including C. elegans and Drosophila. In C. elegans, we showed that the enzyme functions to chemically cross-link tyrosine residues in collagen and other extracellular matrix proteins, thus stabilizing the structure of the cuticle. We will test the hypothesis that a general function of Duox enzymes throughout the animal kingdom is the chemical modification of tyrosine and/or other residues of extracellular matrix. Genetic and biochemical methods will be used to test this hypothesis in Drosophila, where we propose that the tyrosine modifications stabilize the structure of the wing. Phage display methods will be used to develop peptide inhibitors of the peroxidase domains of h-Duoxl and h-Duox2, and these will be used to investigate normal biological functions of Duox such as thyroid hormone biosynthesis and innate immunity. Enzymatic properties and regulation by calcium of Duox will be documented, and the transmembrane topology of the domains will be explored. These studies will provide for the first time fundamental information regarding the enzymology, topology and biological functions of this newly discovered group of enzymes and will document their ability to chemically modify extracellular matrix (ECM). Because ECM is a critical determinant of transformation Duox enzymes may play an important role in cancer biology in some tissues.